The purpose of bloodgroup assays and antibody examination in clinical work is often to obtain compatible erythrocyte preparations for transfusion. The invention concerns the detection and identification of antibodies and antigens and can be used for bloodgroup assay, antibody screening and identification, and performing cross tests. The test is based not on hemagglutination but on the solid-phase principle, without involving washing steps.
In a bloodgroup, specific antigenic determinants on the cell membrane are involved. The information for expressing bloodgroup antigens is fixed on a gene level and heritable. Bloodgroup antigens can give rise to the formation of antibodies. Specific antibodies to bloodgroup antigens are mostly formed after immunization with the corresponding antigen. An exception to this is formed by the so-called "naturally occurring" antibodies which without apparent immunization are demonstrable in the serum (anti-A and anti-B). The presence of antibodies plays an important role in blood transfusion, pregnancy and auto-immunity.
Antibodies can be classified in different ways:
1. xeno-antibodies, allo-antibodies and auto-antibodies; PA1 2. regular and irregular antibodies; PA1 3. naturally occurring antibodies; PA1 4. complete (IgM) antibodies and incomplete (IgG) antibodies. PA1 (i) a reagent containing an analyte binding partner which, in the case where the analyte is a bloodgroup antigen present on erythrocytes, is an antibody capable of binding to the bloodgroup antigen, and, in the case where the analyte is an antibody binding to a bloodgroup antigen, is the bloodgroup antigen present on erythrocytes, PA1 (ii) a reaction vessel comprising an incubation zone, an immobilization zone and a collection zone, with an immunoglobulin-binding substance immobilized in the immobilization zone, and PA1 (iii) a separation medium with a density lower than the density of erythrocytes but higher than the density of an antibody-containing liquid.
Blood transfusion reactions which are induced by allo-antibodies to erythrocytes are called hemolytic transfusion reactions because they are mostly accompanied by an often very strongly accelerated breakdown of erythrocytes. The point is therefore to prevent hemolytic transfusion reactions by careful bloodgroup examination.
Bloodgroup antibodies are nearly always immunoglobulins of the IgG type or the IgM type. The antigen-antibody interaction is dependent inter alia on ionic bonding, hydrogen bridges and hydrophobic effects (displacement of water). The strength of a binding between a binding site of an antibody and an epitope is designated as `affinity`. Antibodies which are able to agglutinate erythrocytes under all conditions are called agglutinins or complete antibodies (mostly IgM). Antibodies which do bind to erythrocytes but give no agglutination (sensitization) are called incomplete antibodies (mostly IgG).
The detection of erythrocyte antigens and corresponding antibodies often takes place by means of agglutination reactions. Agglutination reactions can be allowed to take place in a physiological salt solution. In practice, this is not always optimal. A number of tests can be rendered more sensitive by using a number of aids, such as the use of medium having a low ionic strength (low ionic medium), proteolytic enzymes (e.g. bromelin, papain or ficin), polycations (e.g. polybrene), macromolecules (e.g. albumin), or polymers (e.g. polyethylene glycol, PEG).
An important and widely applied test is the antiglobulin or Coombs test. The antiglobulin test is based on the principle that erythrocytes loaded with, for instance, antibodies of the IgG type can be agglutinated by antiglobulin serum. This is the most important test for demonstrating incomplete antibodies. The antiglobulin test was described by Moreschi in 1908 (Zbl. Bakt 46: 49) and reintroduced in 1945 by Coombs et al. (Lancet 2: 15, Brit. J. Exp. Path. 26: 255). The test can be distinguished into three phases. The first phase is the sensitization phase. During this phase, antibodies bind to the corresponding antigen structures on the erythrocytes (sensitization of erythrocytes). When the binding is optimal, the second phase, viz. the washing phase, takes place. In this phase, all non-bound antibodies are removed from the incubation mixture. Insufficient removal of non-bound antibodies can lead to inactivation of the antiglobulin serum in that these antibodies bind to the antiglobulin. The third phase is the antiglobulin phase, in which antiglobulin is added to the washed sensitized cells, so that the sensitized cells are coupled to each other (agglutination of the erythrocytes).
There is a large variety of serological tests. The most important techniques at present are the tube method, the column test and tests in microtiter plates. A distinction can be made between techniques that are based on hemagglutination and techniques that are based on the solid-phase principle.
a. Tests based on agglutination:
The tube test is a widely used test which also allows prolonged incubations with antibodies. After the reaction with antibodies, the erythrocytes can be settled or be centrifuged to accelerate the agglutination reaction.
In performing the Coombs test, it is necessary, before adding the antiglobulin serum, to wash very thoroughly and frequently. The antigen-antibody reaction is evaluated by gently tapping the tube and then rotating the tube (tip and roll) so that any agglutinates that have formed are dislodged from the tube. The reading of the test must be done promptly by an experienced person and the result of the test cannot be preserved. Because the reading of the test occurs manually, its reproducibility is not optimal either. The disadvantage of this method is that it is difficult to automate.
As indicated, the washing step in the Coombs test takes up much time. Graham et al. (Transfusion 1982, 22: 408; P. L. Mollison, Blood transfusion in clinical medicine, Blackwell, Oxford, 1983, p. 512) developed a new principle which rendered the washing step redundant. This principle was used by Ortho Diagnostic Systems Inc. in a test system (Simwash). According to this system, erythrocytes are separated from the serum by means of a centrifugation step. The separation is based on the fact that the specific gravity of serum (1.03) is lower than that of erythrocytes (1.09). Now, if a mixture of cells and serum is applied on top of a layer of medium of a density between the density of the cells and that of the serum, the erythrocytes will be separated by a centrifugation step from the serum (containing the non-bound antibodies). Thus the erythrocytes are centrifuged from the original incubation mixture. In this way a triple or quadruple washing step is reduced to a single centrifugation step. Then the sensitized cells can be incubated with antiglobulin serum. However, the system proved not to be sensitive enough.
A further simplification of serological tests has been carried through by centrifuging the erythrocytes through a column of (Sephadex) gel or glass beads. The use of transparent, inert, solid particles for distinguishing agglutinates and non-agglutinates was already described by Dalton et al. in 1970 (Becton Dickinson & Co., U.S. Pat. No. 3,492,396). The system whereby gel material is used for the detection of erythrocyte-antibody reactions was described by LaPierre et al. (Transfusion 1990; 30: 109-113, European Patents 0 194 212 and 0 305 337). This system combines the principles of Simwash and the use of solid inert particles to discriminate between agglutinates and non-agglutinates. In this test system, use is made of small columns filled with Sephadex gel. Use can be made of columns not containing any antibodies (e.g. for reverse ABO typing). As a second possibility, a gel column can also contain antibodies which are directed to certain erythrocyte antigens (e.g. for typing). For the antiglobulin test, use is made of gel columns which contain antiglobulin serum. After an incubation the gel columns are centrifuged. In the case of a negative reaction, all erythrocytes will end up at the bottom of the tube; if the test is positive, the erythrocyte agglutinates will be caught on top of or in the gel. In the case of weak reactions, erythrocytes will sediment partly. A major advantage of this test is that in case of the antiglobulin test no washing steps are needed anymore. In fact, during the centrifugation a separation of erythrocytes and serum or plasma constituents takes place. A second advantage is the fact that the results of the test can be properly fixed. The test described is used by DiaMed AG (DiaMed-ID Microtyping System) and Diagast Laboratoires (Chromatest). A comparable system, Ortho Biovue System (Ortho Diagnostic Systems), utilizes glass beads instead of gel material as inert material to catch agglutinates which have formed. A disadvantage of these tests is that a special centrifuge is required for the correct performance of the test. Also, automatic reading of the test requires special reading equipment.
b. Tests based on the solid-phase principle:
Another approach is the use of the solid-phase principle as an alternative to direct and indirect agglutination reactions for bloodgroup assay, antibody screening, antibody identification and cross tests. Application and advantages of the use of solid-phase techniques in the areas mentioned have been described by Rosenfield (Abstracts, 15th Cong. Int. Soc. Blood Trans., Paris pp. 27-33, 1976; U.S. Pat. No. 4,275,053, 1981). Here, inter alia erythrocytes were used which were coupled to the surface of plastic tubes. More recently, systems have been described by Plapp et al. (Am. J. Clin. Path. 82: 719-721, 1984), Bayer et al. (U.S. Pat. No. 4,608,246, 1986), Rachel et al. (Transfusion 25: 24-26, 1985), Plapp et al. (The Lancet 1465-1466, 1986) and Uthemann et al. (U.S. Pat. No. 4,925,786, EP 363 510 and Transfusion 30: 114-116, 1990).
Microtiter plates in combination with the solid-phase principle are employed by, among others, Biotest AG (Solidscreen II for antibody diagnostics), Immucor Inc. (Immunocapture Capture-R systems for antibody screening and identification) and CLB (Microtype for typing erythrocyte antigens). The Capture-R system utilizes a solid phase to which membranes of erythrocytes are bound. During the incubation with serum or plasma from donors or patients, erythrocyte-specific antibodies can bind to these membranes. After the washing phase, whereby the non-bound antibodies are removed, indicator erythrocytes are added. Indicator erythrocytes are sensitized erythrocytes to which anti-human immunoglobulin is bound. Instead, it is also possible to use synthetic particles (spheres) to which anti-human immunoglobulin is bound. If in the plasma or serum antibodies were present, bridging will arise between the antibody bound to the solid phase and the indicator erythrocytes. This principle has also been described by Bayer et al. (U.S. Pat. No. 4,608,246) for antibody screening and identification. In addition to this system, Bayer et al. also describe the performance of a cross test, in which use is made of anti-IgG coupled to the solid phase. Both unknown blood components (plasma and erythrocytes) are put together in the well so that sensitization of the erythrocytes can take place. However, in this set-up, inactivation of the anti-IgG bound to the solid phase will arise and sensitized cells will not be able to bind to the solid phase anymore, unless after the sensitization phase first a washing phase is carried out. Recently, Llopis et al. have described a variant of the solid phase test, intended for screening and identifying antibodies (Vox Sanguinis 1996; 70: 152-156). In this method a monolayer of test erythrocytes (erythrocytes with a known antigen composition) is made in a U-bottom well of a microtiter plate, whereafter incubation takes place with serum which includes antibodies. This is followed by five manual washing steps. The detection takes place by addition of polyspecific anti-human globulin (IgG+C3d) and indicator erythrocytes (these are, in this case, erythrocytes which are sensitized with anti-D antibodies). Finally, a centrifugation step takes place again. Disadvantages of this test are, among others, the limited life of the monolayer of test erythrocytes and the repeated manual washing of the monolayer after incubation with antibodies.
Solidscreen of Biotest AG (antibody screening and identification) and Microtype of CLB (antigen typing) utilize a solid phase to which (inter alia) human IgG is bound. In the wells the incubation of erythrocytes and plasma, serum or antibody-containing reagent takes place. If corresponding antibodies are present, sensitization of erythrocytes takes place. The sensitized cells are then washed to remove non-bound antibodies. Detection takes place by addition of anti-human globulin serum and a centrifugation step, so that the sensitized cells are bound via the anti-globulin to the IgG on the solid phase. In these systems, positive reactions are characterized by a monolayer of erythrocytes on the solid phase. In the case of negative results, no monolayer is formed, but the cells form a so-called "button" in the U-bottom microtiter plate well.
A disadvantage of the known solid-phase microtiter plate tests is that frequent washing is required in order to remove the non-bound antibodies. This washing typically proceeds manually, entailing the continued possibility that patient serum or plasma (potentially infectious material) is spread during the washing steps. A cycle from the washing phase comprises in succession: centrifugation, removal supernatant, addition of medium and resuspension of the erythrocytes. This time-consuming cycle must be repeated a number of times in order to remove all non-bound antibodies. Automation of this washing procedure is possible. This involves one centrifugation step, whereafter supernatant medium is carefully removed, whereafter an amount of medium is applied to the pellet of erythrocytes, in such a manner that no resuspension of the erythrocytes take place. This step must be repeated about six times. Normal microtiter plate washing devices (such as ELISA washers) are not suitable for this washing step. For that purpose, a special washing device is needed. After the washing phase all washing liquid is removed and antiglobulin serum is added. Then the erythrocytes are resuspended and the microtiter plate is centrifuged. The sensitized erythrocytes will be bound to the IgG of the solid phase through the antiglobulin. Non-sensitized erythrocytes will not be able to bind to the solid phase and will be found as button in the U-bottom well. It goes without saying that incomplete washing away of antibodies leads to inactivation of the antiglobulin, so that sensitized erythrocytes cannot bind via the antiglobulin to the solid phase anymore, and will result in a so-called false-negative reaction. The advantage is that automatic processing of results is possible. An important point in this connection is that during the washing phase no loss or only slight loss of cells may arise, since this can present problems when (automatically) reading the test.
The solid-phase tests are not limited to microtiter plates. Pernell (Sanofi Pasteur, EP 0 594 506) describes an affinity gel test in which an immunoglobulin-binding substance (such as protein A, protein G or anti-human globulin) is immobilized on the gel. Incubation of erythrocytes and antibodies occurs above the gel. After this incubation phase centrifugation of the gel column takes place. If antibodies are bound to the erythrocytes, these sensitized erythrocytes will bind to the immunoglobulin-binding gel material and will hence bind to the upper part of the gel column. Cells to which no antibodies are bound will not be bound and end up on the bottom of the column. This principle of binding of sensitized cells to a solid-phase has previously been described by Pharmacia (Cell Affinity Chromatography; Uppsala, Sweden; 1984). Pharmacia therein describes the purification of erythrocytes on the basis of the presence or absence of certain bloodgroup antigens using protein A sepharose 6 MB. Erythrocytes with the A antigen on the surface (bloodgroup A erythrocytes) to which anti-A antibodies are bound can be separated, by binding to the gel material mentioned (via protein A), from erythrocytes which do not possess the A-antigen. The test described by Sanofi Pasteur is therefore a combination of this principle and the centrifugation step for separation of erythrocytes which may or may not be sensitized and non-bound antibodies as used in Simwash (Ortho) and different column systems (Diagast, DiaMed and Ortho). A disadvantage of the test as described by Sanofi Pasteur, where the ligands described are protein G or an antiglobulin, is that no complement factors are bound, as protein G only binds IgG antibodies. In serology, however, erythrocytes loaded with complement factors are also of interest (for instance in demonstrating complement-binding antibodies such as anti-Jk antibodies). In the Sanofi Pasteur gel test, therefore, if these antibodies are to be demonstrated with a high sensitivity, anti-complement too will have to be immobilized on the gel material. The disadvantage of the use of protein A as immunoglobulin-binding material is that antibodies of the type IgG3 cannot be demonstrated (as protein A only binds the IgG subclasses IgG1, IgG2 and IgG4). These can be clinically relevant, however.
Another variant of a solid-phase test has recently been described by Den Boer et al. (WO 95/30904). In this test, which is suitable, for example, for antibody screening and antibody identification, the solid phase is a porous membrane on which an immunoglobulin-binding substance is immobilized. This membrane, which contains pores sufficiently large to pass erythrocytes, is placed in a tube or cup (of, for instance, a microtiter plate). The membrane is disposed in a medium of high density. Incubation of, for instance, erythrocytes and an antibody-containing sample takes place above this medium, in such a manner that no contact can occur between the antibody-containing incubation mixture and the solid phase. After the incubation phase referred to, a centrifugation step takes place. The antibody-containing sample remains above the medium, erythrocytes will be centrifuged downwards. Thus a separation of erythrocytes and non-bound antibodies is achieved. Due to the centrifugation step, the erythrocytes will move to the solid phase, the sensitized erythrocytes will bind to it, while non-sensitized ones will pass through the solid phase. A disadvantage of this test is that reading must be done at two different levels, as the non-sensitized cells are on the bottom of the test system whilst the sensitized cells are on the solid phase which is at some distance above the bottom of the test system.